论文部分内容阅读
目的采用Illumina高通量测序技术和实时荧光定量PCR(Real-Time PCR,RT-PCR)法研究何首乌(PM)致肝损伤与肠道微生物组间的关系,并验证两种定量方法的一致性。方法雄性SD大鼠随机分为5组:对照组、脂多糖(LPS)组、LPS+对乙酰氨基酚(APAP)组、PM组和LPS+PM组;大鼠尾iv给予4.0 mg/kg LPS建立肝损伤模型,各组相应每天1次ig给予0.625 g/kg APAP和12 g生药/kg PM,记录大鼠体质量;分别于造模后2、14 h、5和8 d,对大鼠粪便中细菌16S r RNA基因的V4高变区进行Illumina高通量测序,根据测序结果得出的差异物种,采用RT-PCR进行验证;取造模后8 d大鼠肝脏组织,HE染色,光学显微镜观察。结果大鼠肝脏病理学检查结果显示,与对照组比较,LPS组大鼠存在肉芽肿,PM组无异常病变;与LPS组比较,LPS+PM大鼠肝细胞出现轻度变性和微小肉芽肿增多,LPS+APAP组可见微小肉芽肿和淋巴细胞浸润。Illumina高通量测序结果提示,与对照组比较,随着PM给药次数增加,单独给予PM的大鼠肠道微生物无显著变化;LPS+PM组表现为肠球菌科和毛螺旋菌科细菌逐渐增加,乳杆菌属细菌减少,且与LPS组有差异;RT-PCR结果显示,与对照组比较,随着PM给药次数的增加,单独给予PM的大鼠肠道微生物无显著变化;LPS+PM组肠球菌科、毛螺旋菌科细菌数显著增加(P<0.05),乳杆菌属细菌数显著减少(P<0.05),且与LPS组比较有显著差异。结论 PM肝损伤大鼠存在不同程度的菌群失衡,且Illumina高通量测序和RT-PCR检测结果具有良好的一致性,但Illumina高通量测序技术可获得更多的微生物信息,更具优势。
Objective To investigate the relationship between liver damage caused by Polygonum multiflorum (PM) and intestinal microflora by using Illumina high-throughput sequencing and Real-Time PCR (RT-PCR), and to verify the consistency of the two quantitative methods . Methods Male Sprague-Dawley rats were randomly divided into 5 groups: control group, lipopolysaccharide (LPS) group, LPS + acetaminophen (APAP) group, PM group and LPS + PM group; Liver injury model, each group corresponding to ig 0.6mg / kg APAP and 12 g crude drug / kg PM once a day, recording the body weight of rats; respectively, at 2,14 h, 5 d and 8 d after modeling, Illumina high-throughput sequencing was performed on the V4 hypervariable region of bacterial 16S r RNA gene, and the differential species were identified by RT-PCR. The liver tissues of HE staining and optical microscope Observed. Results The results of pathological examination in liver of rats showed that there were granulomas in LPS group and no abnormal changes in PM group compared with those in control group. Compared with LPS group, mild degeneration and small granuloma were found in hepatocytes of LPS + PM rats , Small granuloma and lymphocyte infiltration in LPS + APAP group. Illumina high-throughput sequencing results showed that, compared with the control group, with the increase in the number of PM administration, intestinal microbiota of rats given PM alone had no significant change; LPS + PM group showed Enterobacteriaceae and Spirillum bacteria gradually The results of RT-PCR showed that there was no significant change in the intestinal microbiota of rats with PM alone as the number of PM administration increased; LPS + The number of bacteria in Enterococcus group and Spirillum sp. In PM group was significantly increased (P <0.05), and the number of bacteria in Lactobacillus was significantly decreased (P <0.05), and there was a significant difference compared with LPS group. Conclusions There is different degree of bacterial imbalance in PM hepatic injury rats, and the results of Illumina high-throughput sequencing and RT-PCR are in good agreement, but Illumina high-throughput sequencing technology can obtain more microbial information and have more advantages .